The bidirectional role of GABAA and GABAB receptors during the differentiation process of neural precursor cells of the subventricular zone

The intricate process of neuronal differentiation integrates multiple signals to induce transcriptional, morphological, and electrophysiological changes that reshape the properties of neural precursor cells during their maturation and migration process. An increasing number of neurotransmitters and biomolecules have been identified as molecular signals that trigger and guide this process. In this sense, taurine, a sulfur-containing, non-essential amino acid widely expressed in the mammal brain, modulates the neuronal differentiation process. In this study, we describe the effect of taurine acting via the ionotropic GABAA receptor and the metabotropic GABAB receptor on the neuronal differentiation and electrophysiological properties of precursor cells derived from the subventricular zone of the mouse brain. Taurine stimulates the number of neurites and favors the dendritic complexity of the neural precursor cells, accompanied by changes in the somatic input resistance and the strength of inward and outward membranal currents. At the pharmacological level, the blockade of GABAA receptors inhibits these effects, whereas the stimulation of GABAB receptors has no positive effects on the taurine-mediated differentiation process. Strikingly, the blockade of the GABAB receptor with CGP533737 stimulates neurite outgrowth, dendritic complexity, and membranal current kinetics of neural precursor cells. The effects of taurine on the differentiation process involve Ca2+ mobilization and the activation of intracellular signaling cascades since chelation of intracellular calcium with BAPTA-AM, and inhibition of the CaMKII, ERK1/2, and Src kinase inhibits the neurite outgrowth of neural precursor cells of the subventricular zone.


Introduction
Adult neurogenesis, the process by which newborn neurons integrate into consolidated neural circuits, has been extensively studied in the subgranular zone of the dentate gyrus and the subventricular zone (SVZ) of the lateral ventricles.Neurons stemming from the subgranular zone merge into the granule cell layer and develop a glutamatergic phenotype, contributing to hippocampal-related functions [1][2][3][4].On the other hand, most of the SVZ-derived neurons undergo a rostral migration that culminates with their integration into the olfactory bulb.These newborn cells develop a GABAergic phenotype and are, indeed, interneurons [5,6] whose function has been linked to accelerating the learning of olfactory discrimination and short-term olfactory memory [7,8].
Morphologically, adult neurogenesis is accompanied by neurite outgrowth and the formation of a multifarious neurite branching rearrangement associated with neuronal maturation [9].Neurite outgrowth is triggered by depolarizing events that favor transient increases in intracellular calcium and the activation of signaling cascades that promote neurite elongation and synapse formation [10].Consistent with this tenet, neuronal precursor cells (NPCs) of the SVZ are stem cells endowed with multiple signaling cascades whose activation converges in the phosphorylation of the cAMP response element-binding protein (CREB), a transcription factor required for neurite outgrowth of NPCs [11,12].
The signaling cascades involved in the differentiation process of NPCs are activated by diverse neurotransmitters, hormones, and biomolecules, including taurine, a free sulfuramino acid found in high concentrations in the mammalian brain [13].It has been well established that taurine has a neurotrophic effect on neurite outgrowth and the thickness of developing neurites [14].In a recent study, we demonstrated that the development of neurite complexity of NPC SVZ mediated by taurine is accompanied by the functional expression of ion channels that reshape the passive electrophysiological properties of NPC SVZ and favor the appearance of active properties, including action potential generation and a Ca 2+ -dependent afterhyperpolarization [15].
Interestingly, a series of studies have documented that NPCs also express GABA B receptors (GABA B R).The blockade of GABA B R promotes hippocampal neurogenesis [16], and in neocortical-derived neurospheres, the blockade of GABA B R favors the proliferation and differentiation process [17].Contrary to these findings, the pharmacological activation of GABA B R has been reported to suppress adult hippocampal neurogenesis [18,19].Despite the possible relevance of GABA B R in the differentiation process of NPCs, the expression of GABA B R on NPC SVZ and its role in the differentiation process of NPC SVZ remains unexplored.
To close this gap, in this study, we explored the interaction of taurine with GABA receptors in the maturation process of NPC SVZ.We first demonstrated the presence of GABA B R in neurospheres of NPC SVZ; then, we explored the role of taurine in the establishment of inward and outward membrane currents that shape the electrophysiological properties associated with neuronal maturation; and finally, we demonstrated the role of several intracellular signaling cascades in the neurite formation of NPC SVZ.Our findings demonstrate that the abundant neuroactive molecule taurine plays a significant role in the neurophysiological maturation of NPC SVZ via an antagonistic modulation of ionotropic and metabotropic GABA receptors acting in a nonsynaptic fashion.

Primary neural precursor cell culture
The experimental procedures were conducted in strict accordance with the Mexican Official Norm for the use and care of laboratory animals "NOM-062-ZOO-1999," the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and with the approval of the Institutional Animal Care and Use Committee of the National University of Mexico (FMED/DI/068/2019, CICUAL018-CIC-2019). Eight-day-old CD1 mice were used to obtain NPC SVZ.The mice were quickly sacrificed by decapitation, their brain was removed, and the SVZ was isolated.Then, the tissue was mechanically dissociated and placed in a DMEM/F12 medium (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12.Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and centrifuged for 5 min at 1000 rpm.The supernatant was removed, and the pellet was resuspended and cultured in a growth medium (DMEM/F12 supplemented with B27, plus epidermal growth factor and fibroblast growth factor 2) to promote progenitor cell proliferation and SVZ neurosphere formation.
The resulting cultures were maintained at 37˚C and 5% CO 2 .After seven days, the primary neurospheres were mechanically disaggregated with trypsin, and a trypsin inhibitor (1:1) was added to stop the reaction.The cells were then cultured in a growth medium for four to five days at 37˚C and 5% CO 2 .For the differentiation process of NPC SVZ, disaggregated cells of the secondary SVZ neurospheres were cultured in 48 wells pretreated with poly-D-lysine (Sigma-Aldrich, St. Louis, MO, USA) and a differentiation medium (DMEM/F12 supplemented with 1% fetal bovine serum (FBS) and complemented with taurine (10 mM)).The taurine dose used in this study was previously determined by performing a dose-response curve [15].
To evaluate the role of the GABA A or GABA B receptor in the differentiation process, the cultured cells were preincubated for one hour with the GABA A receptor antagonist picrotoxin (100 μM), the GABA B receptor antagonist CGP55845 (5 μM), or the GABA B receptor agonist baclofen (100 μM).To determine the participation of different signaling cascades in the differentiation process induced by taurine through GABA receptors, the cells were cultured for differentiation for three days and preincubated with BAPTA-AM (100 μM) in a Krebs-Ringer Buffer without calcium for 30 min.Then, KN93 (10 μM), FR180204 (10 μM), and SrcI (1 μM) were co-incubated in the presence of taurine 10 (mM) for seven days.All the drugs were purchased from Sigma-Aldrich, St. Louis, MO, USA.

Quantitative analysis for cell markers
Microphotographs were obtained with the cell imaging multi-mode plate reader Cytation™ 5 (BioTek Instruments Inc., Winooski, VT, USA) using a green fluorescent protein (GFP) filter cube (excitation 469/35 nm; emission 525/39 nm; dichroic mirror 497 nm) and 465 nm LED; a Texas Red filter cube (excitation 586/15 nm; emission 647/67 nm; dichroic mirror 605 nm) and 590 nm LED; and a 4 0 ,6-diamidino-2-phenylindole (DAPI) filter cube (excitation 377/50 nm; emission 447/60 nm; dichroic mirror 409) and 365 nm LED.Microphotographs of five fields were randomly selected from each coverslip, and the number of total (DAPI positive) cells and cells positive for the different markers in the selected fields were quantified.Cell images were processed using Gen5™ software (BioTek Instruments Inc.).The results were expressed as the percentage of positive cells out of the total number of cells.

Morphometric analysis of dendritic arborization of DCX-and MAP2-positive cells
Microphotographs of DCX-positive (DCX+) cells were randomly obtained with a 20x magnification lens.DCX+ cells were analyzed for each experimental condition.The number of primary, secondary, and tertiary neurites and the length of the primary neurites were determined with Gen 5 3.04.The difference between primary, secondary, and tertiary neurites was analyzed in the different experimental conditions.Manual quantification of dendritic arborization was performed by counting the number of primary dendrites for each neuron using ImageJ software [20] with the Sholl analysis plug-in v3.4.2 [21].Primary neurites were defined as those that originated directly from the soma; secondary and tertiary neurites were defined as those that originated from primary and secondary neurites, respectively.Researchers were blinded to experimental groups prior to performing all morphometric analyses.

Whole-cell patch-clamp recordings
Dissociated NPC SVZ from SVZ neurospheres were taken from a petri dish containing the cell culture, mounted on a coverslip, and transferred to a submersion recording chamber.They were visualized with infrared differential interference contrast optics coupled to a Nikon FN1 microscope (Nikon Corporation, Minato, Tokyo, Japan).The NPC-SVZ were continuously perfused with Ringer's solution at the rate of 3-4 mL/min -1 with the help of a peristaltic pump (120S, Watson-Marlow, Wilmington, MA, USA).The composition of the bath solution was as follows (mM): 132 NaCl, 3.6 KCl, 1.5 MgCl 2 , 2.5 CaCl 2 , 10 HEPES, and 10 D-glucose; pH = 7.30-7.40;osmolarity = 280-290 mOsm.The recording chamber temperature was maintained at 32 ± 1˚C with a single channel temperature controller (TC-324C, Warner Instruments, Hamden, CT, USA).The patch pipettes were pulled from borosilicate glasses using a micropipette puller (P97, Sutter Instruments, Novato, CA, USA).The puller was programmed to obtain pipettes with a tip resistance of 3-5 MO when filled with an intracellular solution containing the following (mM): 140 KCl, 1.1 EGTA, 10 HEPES, 3 Mg 2+ -ATP, 0.3 Na + -GTP, and pH = 7.20-7.30.Whole-cell recordings were performed with an Axopatch 200B amplifier (Molecular Devices, San Jose ´, CA, USA).Acquired data were digitized at a sampling frequency of 20 kHz and filtered at 2 kHz with a Digidata 1322A (Axon Instruments, Palo Alto, CA, USA).Signals were acquired and analyzed offline with pCLAMP 10.6 software (Molecular Devices).

Determination of intrinsic properties
The resting membrane potential (RMP) was determined after the initial membrane break-in from giga-seal to whole-cell configuration in voltage clamp mode.After 2-3 min. of stabilization, a series of negative and positive voltage commands from -100 to +50 (500 ms, 250 pA increments) were injected to determine the input resistance (R N ) and membrane time constant (τ memb ).The R N was calculated as the slope value of a first-order polynomial function f(x) = m x + b fitted to the current-voltage relationship near RMP.The τ memb was determined by fitting a mono-exponential decay function to a voltage response elicited by injecting a negative current step (1 s, −30 pA).The membrane capacitance (C m ) was calculated as the ratio of τ memb to R N , as previously reported [22].

Determination of macroscopic currents
Whole-cell voltage-clamp configuration was performed to evoke total membrane currents by the injection of depolarizing pulses (−90 to +60 mV, 10-mV steps, 500 ms) from a holding potential of −70 mV.The P/4 protocol was employed to subtract leak currents online, and the recording quality was monitored online by incorporating the following criteria: holding currents < 50 pA, stable RMP, and access resistance (< 15 MW, < 20% drift).To generate I-V plots, the current amplitude was measured either at the maximum peak amplitude or as the average over a 100-msec interval at the steady-state current.

Statistical analysis
No statistical method was used to determine the sample size.The statistical analysis was performed with GraphPad Prism 8.The graphs represent the mean ± the standard error of the mean (SEM).The data normality was previously determined with a Kolmogorov-Smirnov test.Most of the data did not meet the normality criteria; and non-parametric tests were applied: Mann-Whitney U for comparing two experimental groups and Kruskal-Wallis for comparing more than two groups.A one-way analysis of variance (ANOVA) test was applied, followed by Tukey's post-hoc test in cases where normal distribution was observed.In cases where the comparison was between two groups, the Student's t-test was applied.In all cases, the accepted level of significance was set to p < 0.05 or a higher statistical significance.

Progenitor markers and GABA B receptor subunits are co-expressed in neural progenitor cells of the subventricular zone Activation of GABA A R with taurine induces neuronal differentiation and neurite outgrowth in NPC SVZ
We previously reported that disaggregated NPC SVZ incubated with taurine (14 days/10 mM) undergoes increased expression of neuronal markers accompanied by the development of passive and active electrophysiological properties [15]  Morphologically, the control cells displayed bipolar-oriented somata with one or two primary neurites; by contrast, the taurine-treated cells exhibited a multipolar morphology with extensive neuritic arborizations and complex branching.Likewise, the number of neurites and intersections was significantly higher in the proximity of the somata of the taurine-treated cells Next, we examined the functional expression of macroscopic currents expressed by the NPC SVZ using the whole-cell patch-clamp technique.Under the voltage-clamp mode, we applied a protocol (see methods for details) that sequentially elicited an inward current followed by an outward current.Taurine incubation dramatically increased the inward current compared to the control cells (inward current in the control condition = −76.2± 24 pA; in the taurine-treated cells = −248.1 ± 36.3 pA; t-test: t (10) = 3.63; p = 0.004; n = 12 for each experimental condition; Fig 2G; blue arrowheads and far-right traces).On the other hand, the outward current exhibited two kinetic components: a fast-inactivating current peak followed by a steady, non-inactivating current that resembles the neuronal I A and the I D potassium currents [22,24].Interestingly, the taurine-treated cells exhibited an increased amplitude of the fastinactivating outward current (fast-inactivating current in the control condition (n = 4) = 1,474.Lastly, the relationship membrane time-constant / somatic input resistance ratio (τ memb /R N ) yielded a membrane capacitance (C m ) of 47.39 ± 9.22 pF for the control and 41.47 ± 14.56 pF (ns) for the taurine-treated cells; t-test: t (10) = 1.02; ns; n = 12 for each experimental condition; suggesting that the observed increase in the current amplitude was unrelated to changes in the somatic area.Next, we corroborated that the pharmacological blockade of GABA A R decreases the neuronal differentiation process mediated by taurine [15].No changes were found in the C m of the taurine + baclofen-treated cells compared to taurine-treated cells (C m in the taurine-treated cells (n = 6) = 48.1 ± 9.22 pF; in taurine + baclofen (n = 3) = 43.84± 11.42 pF; t-test: t (7) = 1.55; ns).Collectively, these results demonstrate that baclofen-induced GABA B R activation does not contribute to the neuronal differentiation process of the NPC SVZ or to the balanced expression of macroscopic currents; on the contrary, GABA B R activation appears to interfere with neurite outgrowth and the functional expression of ion channels.

A rise in intracellular calcium is necessary for the neuronal differentiation process mediated by taurine
Because taurine is a well-known modulator of intracellular Ca 2+ homeostasis (Wu and Prentice, 2010) we explored if calcium mobilization is involved in the morphogenic process of NPC SVZ mediated by taurine.To test this hypothesis, the cultures were preincubated with taurine + BAP-TA-AM.As expected, the intracellular Ca 2+ chelation with BAPTA-AM significantly reduced the number of neurites mediated by taurine (total number of neurites in the presence of taurine We also performed an immunoreactivity assay against the microtubule-associated protein 2 (MAP2) in the cultures exposed to taurine + BAPTA-AM.Immunoreactivity to MAP2 is a strong indicator of stable neuronal morphology during neuronal maturation.In addition to affecting neurite outgrowth, we hypothesized that Ca 2+ chelation with BAPTA would negatively affect neuronal maturation.As expected, BAPTA-AM decreased the MAP2 immunoreactivity (MAP2+ immunoreactivity in the presence of taurine compared to the control cells = 134.6 ± 13.3; p = 0.0525; ns; in the presence of taurine + BAPTA-AM = 69.90± 8.31%; p < 0.001; one-way ANOVA; Tukey's test; F (2, 15) = 23.53;n = 6 for each experimental condition; Fig 6E).Collectively, these experiments demonstrate that the differentiation and maturation process of NPC SVZ mediated by taurine involves intracellular calcium mobilization.results demonstrate that CaMKII, ERK1/2, and the Src kinase play a crucial role in the taurinemediated neuronal differentiation process.More specifically, inhibition of these signaling cascades negatively impacts the morphology and complexity of the differentiation process of the NPC SVZ.

Discussion
The results of this study are summarized as follows: We have provided evidence that taurine, by interacting with GABA A -or GABA B receptors, controls the differentiation process, neurite formation, and functional expression of ionic currents of NPC SVZ.First, we demonstrated the presence of the GABA B receptor subunits, GABA B R1 and GABA B R2, in NPC SVZ derived from neurospheres immunopositive to nestin and Ki-67, which are progenitor and proliferation cell markers, respectively.To the best of our knowledge, this is the first demonstration of the expression of GABA B R on NPC SVZ.Then, we demonstrated that taurine increases the number of DCX+ cells, favors neurite outgrowth, and promotes the development of secondary, tertiary, and higher-order neurites.These morphological changes are accompanied by the functional expression of ionic channels mediating inward and outward currents that differentially respond to the activation or blockade of GABA receptors.Since the entire maturation process was sensitive to PTX, it was mediated by the activation of GABA A R [15].We also showed that pharmacological activation of GABA B R in the presence of taurine does not promote neurite outgrowth, neurite development, or functional expression of ion channels.
Contrary to this finding, the blockade of GABA B R with its specific antagonist CGP 35348 promoted neurite branching and stimulated the development of secondary, tertiary, and higher-order neurites of disaggregated NPC SVZ.We also demonstrated that the taurine interaction with GABA receptors involves an intracellular calcium rise that activates different signaling cascades that stimulate neurite outgrowth.Collectively, these findings show that ionotropic and metabotropic GABA receptors play bidirectional roles in the differentiation and maturation process of NPC SVZ stimulated with taurine.

Role of GABA A R in the differentiation process of NPC SVZ
The role of GABA in the proliferation, differentiation, and maturation process of NPC SVZ and NPCs of the subgranular zone of the dentate gyrus has been well documented.In both neuronal niches, NPCs express the synthesizing enzyme for GABA, glutamate decarboxylase, several GABA transporters, and, in a spatiotemporal manner, GABA A R [23,25,26].In the SVZ, GABA is released in a nonsynaptic fashion, and its binding to the GABA A R triggers depolarization of NPCs [23,26,27].Central to this depolarization process is the inverse chloride gradient during early neuronal development.
It is well documented that the extrusion of intracellular chloride mediated by GABA binding to the GABA A R leads to membrane depolarization with the subsequent activation of different voltage-sensitive channels [28,29], including L-type calcium channels [30][31][32].A Ca 2+ influx via L-type channels activates calcium-dependent signaling cascades, including kinases and nuclear factors that control gene expression and cell fate.Consistent with this activity, neurons differentiated from NPCs express the Ca 2+ -calmodulin-dependent protein kinase II (CaMKII) [33].Activation of CaMKII phosphorylates the cellular transcription factor, the cAMP response element-binding protein (CREB) that, in turn, controls the transcription of genes critical for neurogenesis, synaptic plasticity, and neuronal survival [34,35].We have provided experimental evidence showing that the blockade of CaMKII with KN-93 inhibits neurite outgrowth and neurite branching, suggesting that calcium elevation via GABA A R activates CaMKII and possibly nuclear CREB.
Consistent with this possibility, CREB promotes the neuronal differentiation of NPCs [36,37].Likewise, the taurine-GABA A R interaction may activate other Ca 2+ -dependent kinases, including members of the non-receptor Src-family kinases (SFKs), a family of tyrosine kinases that regulate cell growth, differentiation, migration, and survival.Src activation phosphorylates the focal adhesion kinase (FAK) that regulates proliferation and survival via CREB phosphorylation [38].The plausible participation of the Src kinases, FAK, and CREB in neurite outgrowth was corroborated in our study since the Src inhibitor hindered neurite development of NPC SVZ.In addition, the activation of this signaling cascade is complemented by the possible activation of PIK 3 by Src, which, in turn, activates AKT, a kinase involved in cytoskeletal remodeling [39], a necessary step for dendrite development.Our experimental observations indicate that the activation of GABA A R with taurine triggers a depolarization that activates calcium-dependent intracellular signaling cascades that favor the development of neurites in NPCs.

GABA B R in the differentiation process of SVZ NPC
Prior research has largely neglected the contribution of GABA B R in the differentiation process of NPCs.For example, neocortical embryonic NPCs express the GABA B R1 and GABA B R2 subunits of the GABA B R, and the pharmacological activation of the GABA B R favors the formation of neocortical-derived neurospheres [17].Likewise, the proliferation and survival of newly born cells depend on GABA B R activation, and the blockade of GABA B R increases adult hippocampal neurogenesis [16].These observations were further corroborated by Giachino et al. [18], who demonstrated the expression of GABA B R in hippocampal NPCs and that blockade of GABA B R increased the neuroblast proliferation and differentiation in vivo.These authors suggested that intracellular signaling via GABA B R inhibits neurogenesis and promotes the quiescence of NPCs [18,19].Consistent with these observations, we found immunoreactivity to the GABA B R1 and GABA B R2 subunits in neurospheres derived from the SVZ.Since GABA B R is a metabotropic receptor, its effects should be ascribed to the dissociation of the G i or G o protein into G iα , G oα, and G βγ subunits of the protein G coupled to the GABA B R complex.The family of G iα/oα proteins, can be grouped in G iα , comprising the G iα1 , G iα2 , and G iα3 subunits, and G oα , comprising the G oα1 and G oα2 splice variants.G αi proteins are recognized for their interaction with G protein-coupled receptors (GPCRs) and to inhibit adenylate cyclase (AC) activation, leading to a decrease in cellular cyclic AMP (cAMP) levels.In such a scenario, after GABA B R activation, the subunit G iα would inhibit the adenylyl cyclase (AC), thus decreasing the levels of cyclic adenosine 3´-5´-monophosphate (cAMP) and protein kinase A activity and decreasing the phosphorylation level of nuclear CREB [40].Likewise, the decreased levels of cAMP interfere with the functionality of the cAMP-dependent ERK/MAPK activity, and the downregulation of these kinases also negatively impacts the phosphorylation levels of CREB.
Consequently, it is reasonable to assume that the blockade of GABA B R with CGP55485 yields a number of positive effects related to neuronal differentiation.First, the inactivation of the G iα subunit may increase the amount of cAMP and PKA activity, which would lead to increased levels of CREB phosphorylation [41,42].This same component may activate MAPK, another potential pathway toward CREB phosphorylation [43].In addition, the blockade of GABA B R would result in transient intracellular Ca 2+ elevation, a phenomenon that would lead to CAMKII activation and, subsequently, CREB phosphorylation [44].These plausible mechanisms reflect the complexity and crosstalk of the signaling cascades mediated by the GABA A -and GABA B -receptors that regulate neuronal differentiation.
It is important to consider that other mechanisms may also be involved in the action of taurine in the differentiation process.For example, taurine regulates glutamate receptors and Taurine binds to the GABA A receptor and promotes chloride extrusion and the blockade of potassium channels, favoring cellular depolarization, the activation of calcium channels, and activation of the calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII) enzyme that promotes phosphorylation of the cellular transcription factor cAMP response element-binding protein (p-CREB).Cellular depolarization also activates the non-receptor tyrosine kinase Src and phosphorylates protein kinase B or Akt, which is involved in the PI3K/AKT/mTOR signaling cascade.The blockade of the G i /G o -linked GABA B R with CGP55485 may increase the concentration of cAMP, possibly leading to increased CREB phosphorylation via PKA activation or MAPK cascades.Furthermore, its coupling to calcium channels through the G βγ subunits of the protein G coupled to GABA B R can allow high intracellular Ca 2+ levels, activating the CAMKII signaling pathway, thus continuing the pathway mentioned with the GABA A receptors that could ultimately induce CREB activation.https://doi.org/10.1371/journal.pone.0305853.g008modulates intracellular Ca 2+ signaling, which are essential for regulating neuronal growth and differentiation [45,46].These mechanisms may contribute to neuronal differentiation but require additional investigation that was beyond the scope of this study.

Modulation of membranal currents by taurine
Another relevant finding of our study is the selective modulation of membranal currents of NPC SVZ in response to taurine treatments.Although previous studies have documented the acute effect of taurine on the membrane potential, R N , and action potential firing of immature and mature neurons, these effects are ascribed to an acute-chloride modulation via GABA and glycine receptors [47][48][49][50][51].This possibility is unlikely in our case because NPC of the SVZ were incubated with taurine for 14 days before any experimental manipulation.One possible explanation for the electrophysiological modulation observed in our study is that taurine, in the long term, favors the adequate assembly of ionic channels rather than acutely modulating chloride conductance to alter the kinetics of the inward and outward currents.We hypothesize that this effect is an additional consequence of the differentiation capacity of taurine; however, this premise requires additional investigation (Fig 8).
Previous studies have demonstrated that the inward current evoked by the protocol used in our study is sensitive to TTX [52], which supports the notion that the taurine-mediated differentiation process of NPC SVZ involves the membranal expression of voltage-gated Na + channels responsible for the action potential generation [15].On the other hand, the kinetics of the outward currents observed in this study resemble the 4-AP-sensitive fast-inactivating K + current and the TEA-sensitive delayed K + current recorded in actual neurons [22,24].Taken together, our results suggest that taurine has the potential to selectively modulate the outward currents that determine the passive properties of excitable cells.Although additional experiments are needed to demonstrate whether taurine has the potential to stimulate the appropriate subunit assembly of ion channels, this study provides evidence that taurine has both morphogenic and electrophysiological properties to control the differentiation and maturation process of neural progenitor cells.

Conclusion
The free amino acid taurine plays a central role in neurite outgrowth and the functional expression of ion channels that determine the passive properties of differentiated cells.Our data show that this effect occurs via GABA receptors and the subsequent activation of signaling cascades expressed by NPC SVZ.Likewise, our data suggest that GABA, acting via the ionotropic or the metabotropic receptors, has opposing effects that control adult neurogenesis in the SVZ.
. The first observation was further investigated in the present study.The upper panels in Fig 2A show representative bright-field (10x) microphotographs of disaggregated NPC SVZ cultures in the control condition and incubated with taurine; the lower panels show immunofluorescence against doublecortin (DCX).Compared to the control condition, the taurine-treated cells exhibited an increased number of DCX+ cells (Mann Whitney test, U = 4; p = 0.0019, n = 8 for each experimental condition, Fig 2B) and increased development of somatic prolongations or neurites (Mann Whitney test, U = 426; p < 0.001; Fig 2C).Next, a Sholl-like morphometric analysis was used to determine the pattern of neurite branching of the disaggregated cells treated with taurine.Digital reconstructions were performed from the bright-field microphotographs, and the resulting images were processed to trace neuronal arborizations.Fig 2F shows representative digital reconstructions of NPC SVZ

Fig 1 .Fig 2 .
Fig 1.Expression of neural precursor cell markers and GABA B receptor subunits in secondary neurospheres derived from the subventricular zone.Microphotographs showing the immunoreactivity to precursor cell markers and GABA B subunits of cultured neurospheres.The secondary neurospheres exhibited a mean ratio of 70 ± 5 um.The nuclei of the proliferative cells within the neurosphere were labeled with DAPI staining.Immunoreactivity signals were detected for A) nestin (red) and B) KI67 (green), indicating the presence of neural progenitor cells and active proliferation, respectively.Likewise, the neurospheres exhibited immunoreactivity to the GABA B receptor subunits, C) GABA B R1 (red), and D) GABA B R2 (red).All the microphotographs were taken with a 20x magnification lens.The scale bar applies for all the panels.https://doi.org/10.1371/journal.pone.0305853.g001
taurine (n = 40) DCX+ cells).D) Sholl-like analysis contrasting the number of neurite intersections in the control condition and the taurine-treated cells.Upper inset: area under the curve (AUC) chart showing the total area obtained from the Sholl-like analysis performed in the control and taurine-treated cells (n = 8 DCX+ cells).E) Cumulative probability chart summarizing the number of neurite intersections in the control cells versus the taurine-treated cells.The right shift of the taurine-treated cells mirrors the increased number of neurite intersections (n = 8 for each experimental condition).F) Upper panels: representative digital reconstructions and concentric rings (distance between rings = 10 μM) used for the Sholl-like morphometric analysis.Bottom panel: examples of the increased number of primary, secondary, and tertiary neurites of the taurine-treated cells.G) Left panel: a patchclamp pipette attached to an NPC SVZ incubated with taurine.Notice the cell body and the neurite outgrowths.Middle panel: representative current traces obtained in a control and a taurine-treated cell.The blue arrowheads indicate the inward current, the black arrowheads indicate the fast-inactivating and the persistent, steady-state outward current.Right panel: magnification of the inward currents, black trace control, red trace, inward current of a taurine-treated cell.Bottom left panel: plot of the voltage dependence and maximal amplitude of the inward current.Middle panel: plot showing the maximal peak amplitude of the fastinactivating outward current.Right panel: plot depicting the amplitude of the non-inactivating steady-state outward current.**p < 0.01; ***p < 0.001 or higher statistical significance.Error bars indicate SEM.https://doi.org/10.1371/journal.pone.0305853.g002 Fig 3A shows bright-field microphotographs and the respective immunofluorescence assay against DCX performed in the control, the taurine-, and the taurine + picrotoxin (PTX, 100 μM)-treated cells.Fig 3B shows the decreased number of DCX+ cells in the presence of PTX (DCX+ cells in the presence of taurine = 117.9± 3.93%; one-way ANOVA; Tukey's test; p < 0.01; in the presence of taurine + PTX = 73.49± 3.93%; one-way ANOVA; Tukey's test; p < 0.001; F (2, 21) = 42.05;n = 8 for each experimental condition).Consistent with this finding, Fig 3C shows a reduced number of neurites in the taurine + PTX condition (total number of neurites in the taurine-treated cells compared to the control cells (n = 43) = 138.3± 8.1%; one-way ANOVA; Tukey's test; p < 0.001; in taurine + PTX (n = 23) vs. the taurine-treated cells (n = 43) = 52.6 ± 7.6%; oneway ANOVA; Tukey's test; p < 0.001; F (2, 106) = 38.00).Likewise, the blockade of GABA A R with PTX substantially decreased the intersections computed in the Sholl-like analysis (Fig 3D and 3F).This finding is summarized in the inset AUC graph (AUC in the presence of taurine = 310.4± 39.42%; one-way ANOVA; Tukey's test; p < 0.001; AUC in the presence of taurine + PTX = 81.25 ± 33.74%; one-way ANOVA; Tukey's test; p < 0.001 F (2, 15) = 60.05;n = 6 for each experimental condition.The cumulative probability chart in Fig 3E shows the distribution of the total number of intersections in the three experimental conditions.Representative examples of the decreased neurite branching in response to the blockade of GABA A R with PTX are shown in the digital reconstructions (Fig 3F).The whole-cell patch clamp recordings revealed a series of changes in the amplitude of the macroscopic currents when GABA A R was blocked.Incubation of taurine + PTX decreased the maximal amplitude of the inward current compared to the taurine-treated cells (inward current in the taurine-treated cells = −-248.09± 36.30pA; in the taurine + PTX-treated cells = −32.42± 11.66 pA; t-test: t (10) = 5.65; p = 0.0002; n = 12 for each experimental condition; Fig 3G; blue arrowheads and far-right traces).On the other hand, both the fast-inactivating and the steady-state outward currents exhibited a decreased amplitude in the presence of PTX (fast-inactivating current in the taurine-treated cells = 2,154.5± 422.03 pA; in the presence of taurine + PTX = 1,057.1 ± 129.8 pA; t-test: t (10) = 4.24; p = 0.001; n = 12 for each experimental condition; steady-state outward current in the taurine-treated cells = 1,557.5± 421 pA; in the presence of taurine + PTX = 617.45± 106.6 pA; t-test: t (10) = 3.49; p = 0.005; n = 12 for each experimental condition; Fig 3G; white arrowheads in the middle panel current traces).In the taurine + PTXtreated cells, the R N did not exhibit a significant change compared to the control cells (R N in the taurine-treated cells (n = 6) = 204.6 ± 18.2 MO; in taurine + PTX (n = 5) = 513.5 ± 114 MO; t-test: t (9) = 2.95; p = 0.01).No changes were found in the C m of the taurine + PTXtreated cells compared to the taurine-treated cells (C m in the taurine-treated cells (n = 6) = 47.39 ± 9.22 pF; in taurine + PTX (n = 5) = 55.10 ± 18.13 pF; t-test: t (9) = 1.84; ns).Collectively, these experiments indicate that activation of the GABA A R is necessary for the morphogenic process, neurite development, and functional expression of ion channels mediated by taurine.Activation of GABA B receptors does not stimulate the differentiation process of NPC SVZBecause secondary neurospheres express both subunits of the GABA B R (see Fig1C and 1D), we next determined the role of the metabotropic GABA B R in the differentiation process of NPC SVZ.The cultures were exposed to the GABA B R agonist baclofen (100 μM) in the presence of taurine (10 mM/14 days), and the expression of DCX was evaluated.Fig 4A shows bright-field microphotographs and the respective immunofluorescence assays against DCX in the control, the taurine-, and the taurine + baclofen-treated cells.Activation of GABA B R with

Fig 3 .
Fig 3. Pharmacological blockade of GABA A receptor inhibits neurite outgrowth of NPC SVZ.A) Bright-field and immunofluorescence assays.Nuclei were stained with DAPI (blue) and DCX (red).Arrowheads show cells with a neuronal-type morphology obtained in the indicated experimental conditions.B) Scatter plot summarizing the percentage of DCX+ cells in the control and in the presence of taurine and taurine + PTX (n = 8 cell cultures for each experimental condition).C) Scatter plot showing the total number of neurites in the indicated experimental conditions.PTX prevented the neurite outgrowth observed stimulated with taurine (control n = 34 cells; taurine n = 38 cells; taurine + PTX n = 27 DCX+ cells).D) Sholl-like analysis with the number of neurite intersections in the indicated experimental conditions.Upper inset: AUC chart summarizing the findings of the Sholl-like analysis (n = 8 DCX+ cells).E) Cumulative probability chart.The left shift of the taurine + PTX-treated cells (blue line) mirrors the decreased number of neurite intersections when GABA A receptors were blocked (control n = 34 cells; taurine n = 38 cells; taurine + PTX n = 27 DCX+ cells).F) Representative digital reconstructions and concentric rings used for the Sholl-like analysis.Notice the decreased number of primary, secondary, and tertiary neurites of the taurine + PTX-treated cells (right panel).G) Upper panel: representative current traces obtained from cells treated with taurine or taurine + PTX.Blue arrowheads indicate the inward currents, black arrowheads the fast-inactivating and the persistent, steady-state outward current.Upper right panel: magnification of the inward current from a taurine-treated cell (red trace) and a taurine + PTX-treated cell (blue trace).Bottom left panel: voltage dependence and maximal amplitude of the inward current.Middle panel: the maximal amplitude of the fastinactivating outward current.Right panel: the steady-state amplitude of the non-inactivating outward current.ns = non-significant statistical difference; **p < 0.01; ***p < 0.001 or higher statistical significance.Error bars indicate SEM.https://doi.org/10.1371/journal.pone.0305853.g003

Fig 4 .Fig 5 .
Fig 4. Activation of the GABA B receptor with baclofen does not stimulate the differentiation process of NPC SVZ.A) Bright-field and immunofluorescence assays microphotographs for DCX in the control cells, taurine (10mM), and taurine + baclofen (100 μM)-treated cells.Nuclei were stained with DAPI (blue) and DCX (red).Arrowheads show cells with a neuronal-type morphology obtained in the indicated conditions.B) Scatter plot showing the percentage of DCX+ cells in the indicated experimental conditions.Stimulation with baclofen did not alter the number of DCX+ cells (n = 8 cell cultures for each (n = 41) for each experimental condition compared to the control cells (n = 40) for each experimental condition = 138.3± 7.5%; p < 0.001; in the presence of taurine + BAPTA-AM (n = 23) for each experimental condition compared to the taurine-treated cells = 35.16± 7.6%.p < 0.001; one-way ANOVA., Tukey's test; F (2, 101) = 29.60;Fig 6A).Likewise, BAPTA-AM decreased the total length of the neurites of the taurine-treated cells (total length of neurites in the presence of taurine (n = 91) = 27.4 ± 1.5 μm; p < 0.001; in the presence of taurine + BAP-TA-AM (n = 29) = 11.5 ± 2 μm; p < 0.001.one-way ANOVA; Tukey's test; F (2, 194) = 20.07;Fig 6B).The effects of BAPTA-AM on the neurite outgrowth are shown in the Sholl-like plots (Fig 6C) and summarized in the AUC graph (AUC in the presence of taurine compared to the control cells = 410.4± 32.44%; p < 0.001; in the presence of taurine + BAPTA-AM compared to taurine cells = 127.1 ± 32.44%; F (2, 15) = 56.17;n = 8 for each experimental condition; Fig 6D).

Fig 7 .
Fig 7. Involvement of signaling cascades in the differentiation process and neurite outgrowth of NPC SVZ mediated by taurine.A, B) Scatter plots showing the total number (A) and total length of neurites (B) of NPC SVZ treated with taurine or taurine + the CaMKII inhibitor KN93, taurine + the ERK1/2 kinase inhibitor FR1805, and taurine + the Src kinase inhibitor Srcl.The blockade of these signaling cascades prevented the neurite outgrowth promoted with taurine (control n = 25 cells; taurine n = 42 cells; taurine + KN93 n = 15 cells; taurine + FR1805 n = 16 cells; taurine + SrcI n = 19 MAP2+ cells).C) Sholl-like plot contrasting the number of neurite intersections in the control condition and the blockade of the indicated signaling cascades (n = 6 MAP2+ cells).D) AUC chart showing the total area obtained from the Sholl-like analysis performed in the control cells, in cells treated with taurine, and following the blockade of the indicated signaling cascades (n = 6 MAP2+ cells for each experimental condition).E) Immunofluorescence assay microphotographs against MAP2 in taurine and taurine + the signaling cascade blockers.The nuclei were stained with DAPI (blue).The blockade of CaMKII, ERK½, or Src kinase reduced the immunoreactivity to MAP2 (n = 6 cell cultures for each experimental condition).**p < 0.01; ***p < 0.001 or higher statistical significance.Error bars indicate SEM.https://doi.org/10.1371/journal.pone.0305853.g007

Fig 8 .
Fig 8. Schematic representation of the possible mechanisms necessary for the differentiation of neural precursor cells of the SVZ mediated by taurine.Taurine binds to the GABA A receptor and promotes chloride extrusion and the blockade of potassium channels, favoring cellular depolarization, the activation of calcium channels, and activation of the calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII) enzyme that promotes phosphorylation of the cellular transcription factor cAMP response element-binding protein (p-CREB).Cellular depolarization also activates the non-receptor tyrosine kinase Src and phosphorylates protein kinase B or Akt, which is involved in the PI3K/AKT/mTOR signaling cascade.The blockade of the G i /G o -linked GABA B R with CGP55485 may increase the concentration of cAMP, possibly leading to increased CREB phosphorylation via PKA activation or MAPK cascades.Furthermore, its coupling to calcium channels through the G βγ subunits of the protein G coupled to GABA B R can allow high intracellular Ca 2+ levels, activating the CAMKII signaling pathway, thus continuing the pathway mentioned with the GABA A receptors that could ultimately induce CREB activation.